High efficiency chemically competent E. coli bacteria (bacterial cells that can be transformed with DNA) are used extensively in the generation of cDNA libraries and the cloning of samples containing small amounts of target sequences. The ability to generate representative cDNA libraries, one in which each mRNA species present in the subject cell is represented in the library, relies on many factors. One of the major factors determining the quality of a cDNA library is the number of clones represented in the library. Using competent bacteria having a high transformation efficiency increases the probability of obtaining rare, under-represented clones in plasmid libraries. Also, when cloning samples containing small amounts of target DNA or cloning the DNA products of complex DNA manipulations such as the DNA products of single or multiple blunt ended ligations, the use of high efficiency bacteria is essential.
Early attempts to achieve transformation of E. coli were unsuccessful and it was generally believed that E. coli was refractory to transformation. However, Mandel and Higa (J. Mol. Bio. 53: 159-162 (1970)) found that treatment with CaCl.sub.2 allowed E. coli bacteria to take up DNA from bacteriophage .lambda.. In 1972, Cohen et al. showed CaCl.sub.2 -treated E. coli bacteria were effective recipients for plasmid DNA (Cohen et al., Proc. Natl. Acad. Sci., 69: 2110-2114 (1972)). Since transformation of E. coli is an essential step or cornerstone in many cloning experiments, it is desirable that it be as efficient as possible (Lui and Rashidbaigi, BioTechniques 8: 21-25 (1990)). Several groups of workers have examined the factors affecting the efficiency of transformation.
Hanahan (J. Mol. Biol. 166: 557-580 (1983), herein incorporated by reference) examined factors that affect the efficiency of transformation, and devised a set of conditions for optimal efficiency (expressed as transformants per .mu.g of DNA added) applicable to most E. coli K12 strains. Typically, efficiencies of 10.sup.7 to 10.sup.9 transformants/.mu.g can be achieved depending on the strain of E. coli and the method used (Liu & Rashidbaigi, BioTechiniques 8: 21-25 (1990), herein incorporated by reference).
Many methods for bacterial transformation are based on the observations of Mandel and Higa (J. Mol. Bio. 53: 159-162 (1970)). Apparently, Mandel and Higa's treatment induces a transient state of "competence" in the recipient bacteria, during which they are able to take up DNAs derived from a variety of sources. Many variations of this basic technique have since been described, often directed toward optimizing the efficiency of transformation of different bacterial strains by plasmids. Bacteria treated according to the original protocol of Mandel and Higa yield 10.sup.5 -10.sup.6 transformed colonies/.mu.g of supercoiled plasmid DNA. This efficiency can be enhanced 100- to 1000-fold by using improved strains of E. coli (Kushner, In: Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering, Elsevier, Amsterdam, pp. 17-23 (1978); Norgard et al., Gene 3:279-292 (1978); Hanahan, J. Mol. Biol. 166: 557-580 (1983)) combinations of divalent cations ((Kushner, In: Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering, Elsevier, Amsterdam, pp. 17-23 (1978)) for longer periods of time (Dagert and Ehrlich, Gene 6: 23-28 (1979)) and treating the bacteria with DMSO (Kushner, In: Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering, Elsevier, Amsterdam, pp. 17-23 (1978)), reducing agents, and hexamminecobalt chloride (Hanahan (J. Mol. Biol. 166: 557-580 (1983).
Incubation of E. coli. in solutions that contain multivalent cations is an important step in the transformation of E. coli. A number of multivalent cations are capable of affecting DNA transformation of E. coli. In addition to calcium cations, manganese, magnesium and barium cations can affect DNA transformation of E. coli and the use of manganese or barium cations rather than calcium cations has lead to higher transformation efficiencies with some strains of E. coli (Taketo, Z. Naturforsch Sect. C 30: 520-522 (1975); Taketo, Z. Naturforsch Sect. C 32: 429-433 (1975); Taketo & Kuno, J. Biochem. 75: 895-904 (1975)). A variety of other compounds affect transformation efficiencies. Organic solvents and sulhydryl reagents can also influence transformation efficiencies (Hanahan (J. Mol. Biol. 166: 557-580 (1983); Kushner, In: Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering, Elsevier, Amsterdam, pp. 17-23 (1978); Jessee, J. A. and Bloom, F. R., U.S. Pat. No. 4,981,797 (1991)).
Incubation of E. coli at temperatures around 0.degree. C., often on ice, in buffers containing multivalent cations is an important step in the production or generation of competent cells of E coli. A rapid heat shock or temperature transition after incubation of the E. coli with target DNA further improves transformation efficiencies (Mandel and Higra, (J. Mol. Bio. 53: 159-162 (1970)). Typically, the solutions containing E. coli and target DNA are transferred from 0.degree. C. to temperatures between 37 and 42.degree. C. for 30 to 120 seconds. The temperature at which E. coli bacteria are grown prior to incubation at 0.degree. C. can also affect transformation efficiency. Growing E. coli bacteria at temperatures between 25 and 30.degree. C. can improve the transformation efficiency of E. coli bacteria compared with E. coli bacteria grown at 37.degree. C. (Jessee, J. A. and Bloom, F. R., U.S. Pat. No. 4,981,797 (1991)). E. coli bacteria that are grown at temperatures between 25 and 30.degree. C., in contrast to 37.degree. C., may require a heat shock at less than 37 to 42.degree. C., or a heat shock of a shorter duration, for optimal results (Jesse and Bloom, U.S. Pat. No. 4,981,797 (1991); Inoue et al. Gene 96:23-28 (1990)).
Transformation efficiency can be affected by the E. coli strain used. The selection of an E. coli strain that is capable of high transformation with the specific competence protocol adopted is an important step in the development of a procedure to produce E. coli bacteria capable of high transformation efficiencies. Different strains can exhibit different transformation efficiencies depending on the competence protocol used. Lui and Rashidbaigi, BioTechniques 8: 21-25 (1990), compared the transformation efficiency of five E. coli strains, HB101, RR1, DH1, SCS1 and JV30 and showed that the transformation efficiencies of these strains varied according to the methodology adopted.
A number of procedures exist for the preparation of competent bacteria and the introduction of DNA into those bacteria. A very simple, moderately efficient transformation procedure for use with E. coli involves re-suspending log-phase bacteria in ice-cold 50 mM calcium chloride at about 10.sup.10 bacteria/ml and keeping them ice-cold for about 30 min. Plasmid DNA (0.1 mg) is then added to a small aliquot (0.2ml) of these now competent bacteria, and the incubation on ice continued for a further 30 min, followed by a heat shock of 2 min at 42.degree. C. The bacteria are then usually transferred to nutrient medium and incubated for some time (30 min to 1 hour) to allow phenotypic properties conferred by the plasmid to be expressed, e.g. antibiotic resistance commonly used as a selectable marker for plasmid-containing cells. Protocols for the production of high efficiency competent bacteria have also been described and many of those protocols are based on the protocols described by Hanahan (J. Mol. Biol. 166: 557-580 (1983).
The F episome is a genetic element that may exist as a free genetic element or become integrated into the bacterial genome. The presence of the F episome, whether in a free or integrated form, has important consequences for the host bacterium. F-positive bacteria exhibit surface appendages called pilli, which provide attachment sites that facilitate the infection of certain RNA and single-stranded DNA viruses. Many E. coli strains have been constructed to contain an F plasmid in order to facilitate the infection of those strains by single-stranded DNA viruses. E. coli strains engineered for this purpose include: JM101 (Messing, In Recombinant DNA: Proceedings of the Third Cleveland Series on Macromolecules, Elsevier, Amsterdam p 143-153 (1981)); JM105, JM107, JM109 and JM110 (Yanish-Perron et al., Gene 33: 103-119 (1985)); TG1 (Gibson, Ph.D. Thesis, Cambridge University, England (1984)); TG2 (Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor p. 4.14 (1989)); XL1-Blue (Bullock et al., BioTechniques 5.4:376-378 (1987)); XS127 and XS101 (Levinson et al., Mol. Appl. Genet. 2:507-517 (1984)); 71/18 (Dente et al., Nucleic Acids Res. 11: 1645-1655 (1983)); KK2186 (Zagursky and Berman, Gene 27:183-191 (1984)); and MV1184 (Viera and Messing, Methods Enzymol. 153: 3-11 (1987)).
Transformation efficiency was not thought to be enhanced by the addition of F' episome genetic material. (Hanahan (J. Mol. Biol. 166: 557-580 (1983); Bullock et al. (1987)). Indeed, the addition of a F' episome to the E. coli strain AG1 produced an E. coli strain (XL1-Blue) with a reduced transformation efficiency (Bullock et al. (1987)). Contrary to this background, Applicants'invention involves the use of F' genetic material to provide modified E. coli having improved transformation efficiency compared with E. coli without F' genetic material.
Another rapid and simple method for introducing genetic material into bacteria is electoporation (Potter, Anal. Biochem. 174: 361-73 (1988)). This technique is based upon the original observation by Zimmerman et al., J. Membr. Biol. 67: 165-82 (1983), that high-voltage electric pulses can induce cell plasma membranes to fuse. Subsequently, it was found that when subjected to electric shock (typically a brief exposure to a voltage gradient of 4000-16000 V/cm), the bacteria take up exogenous DNA from the suspending solution, apparently through holes momentarily created in the plasma membrane. A proportion of these bacteria become stably transformed and can be selected if a suitable marker gene is carried on the transforming DNA transformed (Newman et al., Mol. Gen. Genetics 197: 195-204 (1982)). With E. coli, electroporation has been found to give plasmid transformation efficiencies of 10.sup.9 -10.sup.10 /.mu.g DNA (Dower et al., Nucleic Acids Res. 16: 6127-6145 (1988)).
Bacterial cells are also susceptible to transformation by liposomes (Old and Primrose, In Principles of Gene Manipulation: An Introduction to Gene Manipulation, Blackwell Science (1995)). A simple transformation system has been developed which makes use of liposomes prepared from cationic lipid (Old and Primrose, (1995)). Small unilamellar (single bilayer) vesicles are produced. DNA in solution spontaneously and efficiently complexes with these liposomes (in contrast to previously employed liposome encapsidation procedures involving non-ionic lipids). The positively-charged liposomes not only complex with DNA, but also bind to bacteria and are efficient in transforming them, probably by fusion with the cells. The use of liposomes as a transformation or transfection system is called lipofection.